【作者】 黄宜兵；

【导师】 张学忠；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp,RGD）三肽是多种胞外基质（Extracellular matrices,ECM）蛋白和血浆蛋白结构中保守的共有的基本成分,也是广泛存在于细胞间识别系统的基本单位,许多病理学过程与不正常的ECM作用相关,因此含RGD的多肽或化合物有望成为治疗一些重要疾病的新制剂。同时,该类肽的粘附性也成为药物设计的一个新靶点。此外,RGD和RGDS由于含有多个极性氨基酸（Arg、Asp和Ser）和一个中性氨基酸（Gly）,是一个研究酶促合成含极性氨基酸亲水小肽颇具代表性的模型。本论文研究内容主要包含两个部分:一、酶法与化学法合成细胞粘附肽RGD、RGDS及其前体目前,关于RGD的合成报道多为传统的化学合成方法,包括液相合成和固相合成,酶法合成RGD的报道很少。我们首次采取酶法化学法相结合的方式合成了两种RGD肽的前体Bz-RGD-（NH₂）₂三肽和Bz-RGDS-NH₂四肽。即先采用了一种新颖的化学法,利用化学基团之间的活性关系,通过巧妙形成的甘氨酸并制备得到产物二肽GD-（NH₂）₂和GDS-NH₂,这种化学方法省去了保护脱保护步骤,而且具有反应条件温和,合成产率高等优点;R的连接则通过酶促动力学控制方式来完成,在此过程中,我们尝试采用不同的催化剂,构建了不同反应体系来合成目标产物,并对酶促合成反应各种影响因素包括溶剂体系、水含量、pH值、温度及反应时间等进行优化,比较不同催化剂在有机溶剂中催化肽键合成的性质差异,探索亲水小肽酶促合成的特点,建立一条高效廉价的RGD（S）的合成路线。从总体来说,脂肪酶催化合成效果是最佳的。脂肪酶没有肽酶活性,不发生产物的二次水解,而且具有与蛋白酶相似的催化机理,因此可以采更多还原

【Abstract】 Many proteins present in extracellar matrix（ECM）and blood plasma,contain a common tripeptide amino acid sequence Arg-Gly-Asp（RGD）as a recognition site for cellular adhesion,spreading and motility of cells. The RGD motif plays a key role in mediating integrin-matrix interaction and signals transmission , which modulate cell-survival and function. RGD-containing peptides as competitive,reversible inhibitors for the binding of adhesive proteins have been widely used to study adhesive interaction between cells and inhibit tumor metastasis,tumor induced angiogenesis and tumor elicited platelet aggregations. RGD peptides not only play a major role as anchoring molecules but also are important in processes like embryogenesis,cell differentiation,immune-response. X₁-RGD-X₂（X₁ =Gly,Asp;X₂ =Ser,Cys,Val,Phe）,which is the further extension of the RGD, have been reported to make these sequences be even more efficient adhesion motifs to integrins of cell membrane. For example,RGDS peptide exhibits additional potent anti-chemotactic and pro-apoptotic effects independently from its anti-adhesive action,likely by entering the cells and directly activating caspase 8 and 9,and lately caspase 3,implying unexpected intracellular actions of the RGD-motif. In addition,it was also reported that the activity of RGDS is improved upon acetylation of the N-terminus at arginine and amidation of serine at the C-terminus（Ac-RGDS-NH₂）.As we know,it is not available to isolate RGD or other biologically active peptides from whole tissue directly. So many researchers tried to更多还原